Identification of macrophage induced genes of Corynebacterium pseudotuberculosis by differential fluorescence induction
Identification of macrophage induced genes of Corynebacterium pseudotuberculosis by differential fluorescence induction
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DOI：
10.1016/j.micinf.2005.05.002
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发表时间：
2005-10-01
影响因子：
5.8
通讯作者：
Moore, R
中科院分区：
文献类型：
--
作者：
McKean, S;Davies, J;Moore, R
假结核棒杆菌是羊干酪样淋巴结炎的病原体。我们基于维多利亚水母的绿色荧光蛋白（gfp）基因的表达，为这种生物体开发了一个启动子报告系统。构建了含有gfp基因的无启动子载体pSM20，并将启动子插入到gfp基因的上游。在转化为假结核杆菌后，可以通过荧光显微镜观察到荧光，并通过流式细胞术测量相对启动子强度。通过测量携带 dnaK 启动子构建体的热休克假结核杆菌的荧光水平，证明了该系统用于测量基因表达变化的有用性。假结核杆菌在 J774 巨噬细胞内的复制可以通过荧光显微镜监测。该系统的建立允许使用差异荧光来识别巨噬细胞感染后表现上调的基因。编码非核糖体肽合成酶和丙酰辅酶A羧化酶β链的基因被鉴定为具有启动子，这些启动子在被假结核杆菌感染巨噬细胞后表现出增强的活性。皇冠版权所有 (c) 2005 由 Elsevier SAS 出版。版权所有。
Corynebacterium pseudotuberculosis is the etiological agent of the sheep disease caseous lymphadenitis. We have developed a promoter reporter system for this organism based on expression of the green fluorescent protein (gfp) gene from Aequorea victoria. A promoterless vector, pSM20, containing the gfp gene was constructed, and promoters were inserted upstream of the gfp gene. Upon transformation into C. pseudotuberculosis, fluorescence could be visualised by fluorescence microscopy, and relative promoter strength measured by flow cytometry. The usefulness of this system for measuring changes in gene expression was demonstrated by measuring fluorescence levels of heat shocked C. pseudotuberculosis carrying a dnaK promoter construct. Replication of C. pseudotuberculosis within J774 macrophages could be monitored by fluorescence microscopy. The establishment of the system allowed the use of differential fluorescence to identify genes that showed up-regulation following macrophage infection. Genes coding for a non-ribosomal peptide synthetase and the beta chain of a propionyl CoA carboxylase were identified as possessing promoters that demonstrated enhanced activity following macrophage infection by C. pseudotuberculosis. Crown Copyright (c) 2005 Published by Elsevier SAS. All rights reserved.